Utilization of microbial resources from Myanmar traditional fermented tea (Laphet-so)

Abstract

From nine samples of Laphet-so, a total of seventy four bacterial isolates and twenty nine yeast isolates were achieved. It was found that all microbial isolates exhibited significant production of indoles plant hormone due to the screening assay. The bacterial isolates H10 (359.81 ± 0.03 µg/mL) and H14 (365.47 ± 2.82 µg/mL) displayed the highest production of indole-related compounds among all microbial isolates. The Salkowski reagent-based IAA assay is responsive the sequential conversion of tryptophan to indolic IAA. However, the precise IAA quantification by high performance liquid chromatography (HPLC) showed that IAA concentration of 6.71 and 568.82 µg/mL were produced by the isolates H10 and H14, respectively. Through analysis of the 16S rRNA gene sequence, the isolate H14 was identified to be Klebsiella aerogenes. The influence of K. aerogenes H14 on in vitro germination of tea, tomato and sunflower seeds was investigated and it was found that the culture broth from K. aerogenes H14 exhibited a significant increase in the rate of germination, vigor index, and development of both the root and shoot compared to untreated samples of tomato and sunflower seeding, but it was failed to stimulate the tea seed germination. Further the study aims to harness the untapped potential of yeast by exploring the utilization of microbial resources found within Laphet-so. A total of eighteen isolates of thermotolerant yeasts were obtained from eight samples of Laphet-so collected from southern Shan state, Myanmar. These isolates demonstrated the tannin tolerance and six isolates were resistant to 5% (w/v) tannin concentration. All eighteen isolates were capable of carboxy-methyl cellulose (CMC) degrading and the DK isolate was only one isolate showing ethanol production at 45˚C noticed by gas formation. This ethanol producing yeast was identified to be Cyberlindnera rhodanensis through the sequence analysis of the D1/D2 domain of the LSU rRNA gene. C. rhodanensis DK produced thermostable extracellular β-glucosidase with the highest total activity 1.70 ± 0.01 U when cultured at 37˚C for 24 h using 0.5% (w/v) CMC as a carbon source. The best carbon sources for extracellular β-glucosidase production were found to be xylan and xylose, with β-glucosidase activity of 3.07-3.08 U/mL achieved when the yeast was cultivated in the yeast malt extract (YM) broth containing either 1% (w/v) xylan or xylose as a sole carbon source, at 37˚C for 48 h. The optimal medium compositions for enzyme production achieved by using Plackett-Burman design and Central Composite Design (CCD) was found to be yeast extract 5.83 g/L, peptone 10.81 g/L and xylose 20.20 g/L, resulting in a production of 7.96 U/mL, while the medium of yeast extract 5.79 g/L, peptone 13.68 g/L and xylan 20.16 g/L gave 9.45 ± 0.03 U/mL for 48 h cultivation at 37˚C. The enzyme was also exhibited remarkable stability under varying temperature conditions such as 100% stable for 3 h at 35˚C, 88% maintained for 3 h at 45˚C and 75% stable and retained for 3 h at 55˚C. The applying of C. rhodanensis DK in fermentation of a healthy alcoholic beverage using cashew apples juice (CAJ) as substrate was investigated. Optimal conditions for sugar-to-ethanol conversion were determined by conducting experiments with glucose concentrations of 5% and 10% (w/v). The ethanol conversion rate was enhanced upon adjusting the pH of CAJ to 6, and fermentation was extended over a 12-days period. To accelerate sugar consumption within 7 days, co-culture fermentation approach was employed utilizing Saccharomyces cerevisiae TISTR 5088 and Lactiplantibacillus pentosus A14-6. All yeast strains demonstrated vigorous growth, with concentrations averaging around 6.8 Log cfu/mL and pH levels ranging from 3.5 and 5.6. Total acidity levels were within the range of 0.02 ± 0.00 to 0.28 ± 0.00 M, while total sugar and reducing sugar levels remained consistent in the range of 2.0 ± 0.00 - 7.7 ± 0.00 g/L and 1.8 ± 0.001 - 6.6 ± 0.00 g/L, respectively, following mono- and co-culture fermentation. The fermented CAJ displayed ethanol contents ranging from 20.5 ± 0.06 - 48.4 ± 0.09 g/L after 7 days of fermentation. Furthermore, the presence of bioactive compounds and the antioxidant activity of the fermented product was significantly increased including total phenolic and total tannins, by a factor of 1.9 ± 0.01 - 2.0 ± 0.01 mg GAE/mL and 1.4 ± 0.04 - 1.5 ± 0.01 mg TAE/mL following the fermentation period. This increase was observed in both mono-culture C. rhodanensis DK and co-culture C. rhodanensis DK + L. pentosus A14-6. Additionally, the fermentation process involving these strains led to a notable rise in the antioxidant activity. The unfermented product initially showed the increase of antioxidant activity from 53.45 ± 0.3% to 65.05 ± 0.22% after the fermentation process. The study highlights the achievement of the highest β-glucosidase enzyme activity of 14.0 ± 0.2 mU/mL, during the co-culture fermentation of C. rhodanensis DK + L. pentosus A14-6. Furthermore, this fermentation combination received significantly high scores in aroma sensory evaluations.